Dental Biofilm and Saliva Biochemical
Composition Changes in Young Orthodontic
Shady Ahmed Moussa1*, Hany Gamei Gobran2, Mohamed Ahmed Salem3
and Ibrahim FaroukBarkat4
lDDS, MRACDS (DPH), Lecture in Pediatric Dentistry and Oral Health, Zagazig University, Egypt, Consultant of Pediatric Dentistry and Dental Public
Health (PHCC, Qatar)
2DDS, Lecture in Oral and Dental Biology, Al-Azhar University, Faculty of Dentistry, Cairo, Egypt
3DDS, Lecture in Orthodontic, Al-Azhar University, Faculty of Dentistry, Assuit, Egypt
4DDS, Lecture in Pediatric Dentistry and Oral Health, Al-Azhar University, Faculty of Dentistry, Cairo, Egypt
Shady Ahmed Moussa,Lecture in Pediatric Dentistry and Oral Health, Zagazig University, Egypt/Consultant of Pediatric Dentistry and Dental Public Health (PHCC, Qatar), P.O. 26555; E-mail: @
Received: February, 14, 2017; Accepted: February 20, 2017; Published: February 28, 2017
Moussa SA, Gobran HG, Salem MA, Barkat IF (2017) Dental Biofilm and Saliva Biochemical Composition Changes in Young Orthodontic Patients. J Dent Oral Disord Ther 5(2): 1-5. DOI: http://dx.doi.org/10.15226/jdodt.2017.00176
Saliva and dental biofilm of children with orthodontic treatment
may be associated with high risk factors that increase incidence of
caries development in this population.
Aim:To assess the dental biofilm and saliva biochemical
composition of young fixed orthodontic patients.
Design:The sample comprised 64 participants between 12
to 18 years of age, of whom 32 had fixed orthodontic treatment as
study group and 32 did not as a control group. Supragingival biofilm
samples were collected from all teeth of all participants by using
sterile curettes. The level of insoluble extracellular polysaccharide
(IEPS)Calcium (Ca), and phosphorus (Pi) concentrations in dental
biofilm was measured using phenol sulphuric acid colorimetric
method. The estimated unstimulated salivary flow, pH, buffering
capacity and count of Streptococcal Mutants were determined on
selective media of all participants
Results:Dental biofilm Ca, and Pi concentration did not differ
between both groups but the dental biofilm of orthodontic patients
showed higher IEPS levels parallel with high salivary count of S.
Mutans. However, there were lowering in pH and buffering capacity
than others without orthodontic treatment (P < 0.05).
Conclusions:The saliva and dental biofilm of fixed orthodontic
patients have higher cariogenic incidence than who are without
Enamel smooth surfaces demineralization, usually is
unfortunately a common complication that range from 2-96%
of orthodontic patients . This variation arises as a result
of different methods used to assess and score the presence
of decalcification . However, the teeth most commonly
affected are maxillary lateral incisors, maxillary canines and
mandibularpremolars . Another study revealed that, any tooth
in the mouth can be affected, and often a number of anterior teeth
show decalcification. Whilst the surface demineralizion remains
intact, there is a possibility of demineralization of the lesion. In
severe cases, frank cavitation is seen which requires restorative
intervention. Half of children treated with fixed orthodontic
appliances had at least one white spot after treatment; maxillary
lateral incisors are most commonly affected site . The incidence
or number of white spot formations did not in proportional to
the length of treatment, although many studies [5,6] found that
rapidly demineralization can occur within thefirst month of fixed
orthodontic appliance treatment. This has focused on aesthetic
implications and the need for the rate of caries assessment
before treatment. On the other hand, there was no incidence of
white spot formation associated with lingual bonded retainers,
which would suggest flow rate and salivary buffering capacity,
have an important role in surface protection against acid attack
. Some authors concluded that, the biochemical of saliva and
dental biofilm contribute significantly to interact between the
oral environment and the mineral tissue of the teeth. Saliva is
an exocrine secretion with 99% water and a variety of proteins
and electrolytes including sodium, potassium, Calcium, chlorine,
magnesium, bicarbonate, and phosphorus. The main functions of
saliva include protecting and cleaning the mouth, also to maintain
the pH and integrity of hard and soft tissues in the oral cavity .
The analysis of organic and inorganic composition of saliva may
contribute to the evaluation of caries incidence and to diagnose
of other associated diseases . Cariogenicity of the dental
biofilm depend on electrolytes level such as fluoride, calcium,
and phosphorus, may also be associated with caries incidence
increase if its Insoluble extracellular polysaccharide content
is low [9,10]. Insoluble extracellular polysaccharide plays an
important role in bacterial adhesion; the Insoluble extracellular
polysaccharide on cell walls of the bacteria also increases biofilm
thickness, resulting in increased acid formation at the tooth
biofilm interface . This study was conducted to evaluate the
changes in biochemical composition of dental biofilm and saliva
in fixed orthodontic young patients.
Material And Methods
According to world medical association declaration of
Helsinki , study population and ethical local institutional
approval for study sample comprised 64 participants between
12 to 18 years-old. The study group was composed of 32 patients
scheduled for fixed orthodontic treatment at the department
of orthodontics, (Al-Azhar University, Assuit, Egypt), and the
control group consisted of 32 patients without any orthodontic
treatment. Patients with debilitating diseases, that were under
current treatment with drugs, that were users of mouth wash,
that had dental prosthesis or inability to provide a saliva sample
due to lack of cooperation or difficulty expectorating were
excluded from the study. All parents or legal guardians received
adequate information and written consent for collection of dental
biofilm and saliva samples from participants.
Dental biofilm samples
Prior collection of samples, patients were asked to stop
oral hygiene measures for 24 hour, which was performed on
other day from the clinical examination. Supragingival biofilm
samples were collected from all teeth by using sterile curettes
and stored at -20° for analysis . For 24 hours, dental biofilm
samples were vacuum-dried over phosphorus pentoxide .
After dissection dry weight of samples were measured using
an analytical balance. For Ca, and Pi extraction, 0.5ml of hcl was
added to the microtube in the proportion of 0.1ml/mg dry weight
biofilm. At room temperature, after 3 hour and under constant
agitation, an equal volume of total ionic strength adjustment
buffer II, modified with 20g naoh/L, pH 5.0 was added to the
microtube. For 10 min, the samples were centrifuged at 11,000g,
and the supernatant was retained for measuring of the Ca, and Pi
concentrations. Ca, Pi level were measured by using (ELI TECH
kit), processed in microlab analyzer spictrophoton (Microlab
300). 1M naoh was added to the remaining precipitate after
washing by saline in the proportion of each 0.2ml/mg dry
weight biofilm. For 3 hours, samples were agitated and then
centrifuged. Supernatant was removed for measuring Insoluble
extracellular polysaccharide (IEPS) levels that were determined
by colorimetric phenol sulphuric acid method [15,16].
Salivary flow rate
At least 2 hours after eating and oral hygiene measures in
order to minimize the effects of diurnal variability in salivary
composition . Unstimulated saliva samples collected from all
the groups by spitting into a preweighed tube during an one min
period which was weighed again, and the unstimulated flow rate
Streptococcal Mutans Count(S. Mutans)
About 3ml from saliva samples which collected were stored
in a sterile calibrated universal container that were divided
into two separate parts of samples, one of them inoculated
onto MitisSalivarius agar media (Becton Dickinson and DIFCO
Company, Chicago, USA)was used for isolation S. Mutans that is
the selective medium  mitisSalivarius agar media contents:
(Pancreatic digest of casein 6gm, Proteose peptone 9gm, Proteose
peptone 5gm, Saccharose 50gm, Dextrose 1gm, Dipotassium
phosphate 4gm, Trypan blue 0.075gm, Crystal violet 0.008gm
and Agar 15gm) after the samples were taken. The medium was
prepared according the manufacturing instructions as: 90gm
of the medium and 150gm sucrose were dissolved in 1liter of
distilled water by heating. The dissolved components were
autoclaved at 121° for 15 minutes and left to cool to 45-50° and
just prior to pouring, 1ml of 1% sterilized potassium tellurite and
1ml of 200 units/ml sterilized Bacitracin were added. Sterilization
of Potassium Tellurite and Bacitracin was performed by filtration
through millipores bacterial filters. About 20ml of the medium
was poured in each Petri plate, all allowed to solidify at room
temperature and then stored in the refrigerator at 4° for no more
than four weeks. Identification of oral S. Mutans was confirmed
by biochemical tests like mannitol and sorbitol fermentation and
catalase  colony counting was done with a magnifying glass
and the count of S. Mutans was expressed as the number of colony
forming units per milliliter (cfu/ml) of saliva. Semiquantitation of
the number of colonies was done by multiplying the actual colony
count with 1x10-5 because of the part that the saliva sample was
diluted one thousand times 1:5 dilution [20,21].
Salivary pH and buffering capacity
Other part of saliva samples used to measure salivary pH
by using pH meter  (pH18 Aqua Lytic Co., USA).Buffering
capacity is determined by quantitative test using a handheld. This
method involves the addition of 0.5ml of saliva to 1.5ml of 5M
Hcl. Mixture was vigorously shaken. Then stream of Nitrogen was
passed through the mixture for 20 minutes to eliminate carbon
dioxide from the sample and allowed to stand for 10 min when
the final pH is measured .
Data were checked entered and analyzed by using SPSS
(version 22). Data were presented as mean and Standard
Deviation (SD) or quantitative variable. Student's t-test was
used for composition of two means (P < 0.05) was considered
statistically significant. Normality was tested using the
Shapiro-Wilk test. (Table 1) shows the characteristics of study
participants. No group differences were found in participant age
and sex (P > 0.05). Since no statistically significant differences
were found between sex and age so that, data were combined for
them. (Table 2) shows nonmicrobialbiofim concentration of Ca,
Pi and salivary flow rate (P > 0.05). These values did not differ
between the two groups. Salivary pH and buffering capacity
were, however, significantly lower in Salivary samples obtained
from study group (P < 0.05). Additionally, IEPS concentrations
were significantly higher (P < 0.05) and S.Mutans colonies count
(P < 0.001) in study group.
Table 1: Shows the mean values, standard deviations and the
statistical analysis of for sex and age.
Significant differences (p < 0.05) *
Insignificant differences (p > 0.05) NS
Sample size calculation: done at 80 power and 95% Cl the estimated
sample is 64 participate 32 in each group using open Epi, (version 2).
Table 2: shows the mean values, standard deviations range and the
statistical analysis of the oral variables.
Calcium (Ca) mg/dl
14.1 ± 1.2
(10.77) - (17.1)
14.1 ± 1.75
(11.1) - (19.3)
Phosphorus (Pi) mg/dl
27.4 ± 2.6
(19.15) - (31.4)
26.2 ± 3.6
(22.06) - (34.21)
187.1 ± 60.9
(118.92) - (327.8)
155 ± 67.1
(101.19) - (331.23)
6.1 ± 0.3
(5.6) - (6.8)
6.5 ± 0.46
(5.54) – (7.14)
2.114 ± 0.184
(1.9) - (2.56)
2.365 ± 0.248
(1.98) - (2.8)
Unstimulated Salivary flow rate
0.693 ± 0.005
(0.604) - (0.791)
0.694 ± 0.04
(0.628) - (0.776)
St. mutans (cfu/ml) ×105/ml
2.02 ± 0.46
(1.337) - (2.767)
1.4 ± 0.5
(0.604) - (1.984)
Significant differences (P < 0.05) *
Highly significant differences (P < 0.001) **
Insignificant differences (p > 0.05) NS
Saliva has an important role in caries development due to
many factors such as it participates in the dilution of substances
intra oral cavity, mechanical cleaning, post-eruptive maturation,
enamel dental remineralization and demineralization, pellicle
formation, antimicrobial action and buffering action produced
by biofilm and foods [24,25,26,27]. Many studies have been
conducted to investigate the changes of microbial environment
in patients undergoing fixed orthodontic treatment [28,29,30].
On the other view, a few previous researches designed to
evaluate changing salivary nonmicrobial composition of
orthodontic patients [31,32]. The saliva of fixed orthodontic
appliances patients had lower pH, buffering capacity and calcium
concentration than that of patients without it. These oral changes
are enough to cause tooth demineralization . The saliva
buffering capacity, defined as salivary resistance to change in pH,
is assigned to carbonate bicarbonate systems, phosphates and
proteins [33,34]. The bicarbonate system is responsible for about
reported that the salivary calcium concentration is highly
dependent on salivary flow rate and pH, so that, the moreincrease
in the salivary flow rate the more increase in the calcium ions
concentration of saliva . However, some other authors have
found that the concentration of calcium ions decreases as salivary
flow rates increase [37,39]. Our finding revealed that, there was
insignificant correlation between calcium ion concentration and
salivary flow rate or between salivary flow rate and buffering
capacity. Some others found [40,41,42] an increase in the
salivary flow rate of fixed orthodontic appliances patients.
Laine and Pienihakkinen  concluded that, the difference was
insignificant between these two variables and also did not find
any correlation. Positive correlation between salivary flow rate
and buffering capacity has been reported by others [26,44]. These
finding were in disagreement with the result of the present study,
since an insignificant correlation between both groups studied
was found.This variation arises as findings attributed to different
methods used to assess and determine levels of these variables.
Teixeira et al,  reported that, there was a significant decrease
in salivary buffering capacity and pH whose may be lead to tooth
demineralization in patients using orthodontic appliances that
support direct relation between pH and buffering capacity of
saliva in decreases and increased risk of caries. These previous
findings were parallel to our results and agreement with result of
Kanaya et al,  results. Therefore, in present study, there was
significant increase of IEPS through study group; Mattos-Granner
et al,  showed that IEPS producing ability of cariogenic
microorganisms may play a greater role in development of caries
rather than the number of microorganisms present. In other
word, the higher concentrations of IEPS the more cariogenicity
. Sucrose rich diets are strongly correlated with IEPS
production in dental biofilm, and increase cariogenic bacteria,
in turn, associated with a greater ability to produce IEPS .
According to Opaloglu-Ak et al.,  long term utilization of
orthodontic appliances may have a negative effect on microbial
flora and increase the risk of new carious lesions. Moreover,
Maret et al.,  showed that oral microflora changed with time
in the orthodontic patients group compared to the control group,
in S. Mutans and Lactobacillus, numbers increased during the
6 months of follow-up in their study and these results concur
present study findings positively.
Although, our finding revealed that the biochemical
composition being similar between both groups, the caries
risk factors were altered in the orthodontic patients So that
the effective preventive measures, oral hygiene measures, diet
counseling, prophylaxis and topical fluoride application should
be applied for orthodontic patients to control increasing in
biofilm cariogenicity which reflect on the prevalence of caries.
Why This Paper Is Important In Dentistry Field?
Dentists should be aware that most of orthodontic
appliances patients from children and adolescents who have
oral environment with higher cariogenic potential than who
are without appliances. Therefore, supervision for restricted
oral hygiene measures by parents or guardians is fundamental
for effective control and the prevention of dental caries in
- Chang HS, Walsh LJ, Freer TJ. Enamel demineralization during orthodontic treatment. Aetiology and prevention. Aus Dent J. 1997;42(5):322-327. doi: 10.1111/j.1834-7819.1997.tb00138.x
- Mitchell L. Decalcification during orthodontic treatment with fixed appliances,An overview.Br J Orthod. 1992;19(3):199-205.
- Geiger AM, Gorelick L, Gwinnett AJ, Griswold PG. The effect of a fluoride program on white spot formation during orthodontic treatment. Am J Orthod Dento Orthop. 1988;93(1):29-37.
- Gorelick L, Geiger AM, Gwinnett AJ. Incidence of white spot formation after bonding and banding. Am J Orthod. 1982;81(2):93-98.
- O'Reilly MM, Featherstone JD. Demineralisation and remineralisation around orthodontic appliances, in vivo study. Am J Orthod Dento Orthop. 1987;92(1):33-40.
- Oggard B, Rolla G, Arends J. Orthodontic appliances and enamel demineralisation. Part 1. Lesion development. Am J Orthod Dento Orthop. 1988;94(1):68-73. doi.org/10.1016/0889-5406(88)90453-2
- IEPStein JB, Scully C. The role of saliva in oral health and the causes and effects of xerostomia. J Can Dent Assoc. 1992;58(3):217-221.
- Kcohler B, Pettersson BM, Bratthall D. Streptococcus mutans in plaque and saliva and the development of caries. Scand J Dent Res. 1981;89(1):19-25.
- Cury JA, Rebello MA, Del Bel Cury AA. In situ relationship between sucrose exposure and the composition of dental plaque. Caries Res. 1997;31(5):356-360.
- Cury JA, Rebelo MA, Del Bel Cury AA, Derbyshire MT, Tabchoury CP. Biochemical composition and cariogenicity of dental plaque formed in the presence of sucrose or glucose and fructose. Caries Res. 2000;34(6):491-497.
- Nisengard RJ, Newman MG. Oral Microbiology and Immunology.2nd edn. Rio de Janeiro: Guanabara Koogan.1994.
- World Medical Association Declaration of Helsinki, Ethical Principles for Medical Research, Involving Human Subjects. J Amer Medi Asso. 2013.
- Nobre dos Santos M, Melo dos Santos L, Francisco SB, Cury JA. Relationship among dental plaque composition, daily sugar exposure and caries in the primary dentition. Caries Res.2002;36(5):347-352.
- Pearce EI, Hancock EM, Gallagher IH. The effect of fluorhydroxyapatite in experimental human dental plaque on its pH, acid production and soluble calcium, phosphate and fluoride levels following glucose challenge. Arch Oral Biol.1984;29(7):521-527.
- Cury JA, Marques AS, Tabchoury CP, Del Bel Cury AA. Composition of dental plaque formed in the presence of sucrose and after its interruption. Braz Dent J.2003;14(3):147-152. doi.org/10.1590/S0103-64402003000300001
- Dubois M, Grilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Analyt Chem. 1956;28(3):350-356. doi: 10.1021/ac60111a017
- Varma S, Banerjee A, Barlett D. An in vivo investigation of associations between saliva properties, caries prevalence and potential lesion activity in an adult UK population. J Dent. 2008;36(4):294-299.doi: 10.1016/j.jdent.2008.01.009
- Gold O, Jordan H, Van-Houte J. A selective medium for S. mutans. Arch Oral Biol. 1973;18(11):1357-1364.
- Beighton D, Russell R, Whiley R. A simple biochemical scheme for the differentiation of Streptococcus mutans and Streptococcus sobrinus. Caries Res. 1991;25(3):174-178.
- McGhee J, Michalek S, Cassell G: Editors. Dental Microbiology Philadelphia: Harper and Row 1982.
- Beighton D. A simplified procedure for estimating the level of Streptococcus mutans in the mouth. Br Dent J. 1986;160:329-330.
- Kitasako Y, Moritsuka M, Foxton R M, Ikeda M, Tagami J, Nomura S. Simplified and quantitative saliva buffer capacity test using a hand-held pH meter. AM J Dentist, 2005;18(3):147-150.
- Moritsuka M, Kitasako Y, Burrow MF, Ikeda M, Tagami J. The pH change after HCl titration into resting and stimulated saliva for a buffering capacity test. Aust Dent J. 2006;51(2):170-174.
- Teixeira HS, Kaulfuss SMO, Ribeiro JS, Pereira BR, Brancher JA, Camargo ES. Calcium, amylase, glucose, total protein concentrations, flow rate, pH and buffering capacity of saliva in patients undergoing orthodontic treatment with fixed appliances. Dent Press J Orthod. 2012;17(2):157-161. doi.org/10.1590/S2176-94512012000200026
- Anderson P, Hector MP, Rampersad MA. Critical pH in resting and stimulated whole saliva in groups of children and adults. Int J Pediatr Dent. 2001;11(4):266-273. doi: 10.1046/j.1365-263X.2001.00293.x
- Ericson T, Mäkinen KK. Saliva formation, composition and possible role. In:Thylstrup A, Fejerskov O.Textbook of cariology. Copenhagen: Munksgaard. 1986;28-45.
- Marsh PD. The significance of maintaining the stability of the natural microflora of the mouth. Br Dent J. 1991;171(6):174-177.
- Bonetti1 G, Parenti S, Garulli G, Gatto M, Checchi L. Effect of fixed orthodontic appliances on salivary properties Progr in Orthod. 2013;14:13.
- Rosenbloom RG, Tinanoff N. Salivary Streptococcus mutans levels in patients before, during and after orthodontic treatment. Am J Orthod Dentofac Orthop. 1991;100(1):35-37. doi: 10.1016/0889-5406(91)70046-Y
- Pandis N, Papaioannou W, Kontou E, Nakou M, Makou M, Eliades T. Salivary Streptococcus mutans levels in patients with conventional and self-ligating brackets. Eur J Orthod. 2010;32(1):94-99. doi: 10.1093/ejo/cjp033
- Peros K, Mestrovic S, Milosevic SA, Slaj M. Salivary microbial and nonmicrobial parameters in children with fixed orthodontic appliances. Angle Orthod. 2011;81(5):901-906. doi: 10.2319/012111-44.1
- Li Y, Hu B, Liu Y, Zhang C, Wang S. The effects of fixed orthodontic appliances on saliva flow rate and saliva electrolyte concentrations. J Oral Rehabil. 2009;36(11):781-785. doi: 10.1111/j.1365-2842.2009.01993.x
- Birkhed D, Heintze U. Salivary secretion rate, buffer capacity and pH. In: Tenovuo JO. Human saliva: clinical chemistry and microbiology. Boca Raton: CRC Press. 1989;1:25-74.
- Newbrun E. Preventing dental caries: current and prospective strategies. J Am Dent Assoc. 1992;123(5):68-73.
- Dowd FJ. Saliva and dental caries. Dent Clin North Am. 1999;43(4):579-597.
- Edgar WM. Saliva: its secretion, composition and functions. Br Dent J. 1992;172(8):305-312.
- EricsonT, Mäkinen KK. Saliva-formation, composition and possible role. In:Thylstrup A, Fejerskov
- Jenkins GN. The physiology and biochemistry of the mouth.4th ed. Oxford: Blackwell Scientific. 1978;284-359.
- Lagerlof F, Oliveby A, Ekstrand J. Physiological factors influencing salivary clearance of sugar and fluoride. J Dent Res.1987;66(2):430-435.
- Chang HG, Walsh LJ, Freer TJ. The effect of orthodontic treatment on salivary flow, pH, buffer capacity, and levels of mutans streptococci and lactobacilli. Aust Orthod J.1999;15(4):229-263.
- Kanaya T, Kaneko N, Amaike C, Fukushima M, Morita S, Miyazaki H, et al. A study on changes in caries risk and microbial flora with the placement of Edgewise appliance. Orthod Waves. 2007;66(2):27-32.
- Ulukapi H, Koray F, Efes B. Monitoring the caries risk of orthodontic patients.Quintessence Int.1997;28(1):27-29.
- Laine M, Pienihakkinen K, Ojanotko-Harri A, Tenovuo J. Effects of low-dose oral contraceptives on female whole saliva. Arch Oral Biol.1991;36(7):549-552.
- Bardow A, Moe D, Nyvad B, Nauntofte B. The buffer capacity and buffer systems of human whole saliva measured without loss of CO2. Arch Oral Biol.2000;45(1):1-12.
- Mattos-Graner RO, Smith DJ, King WF, Mayer MP. Water-insoluble glucan synthesis by mutans streptococcal strains correlates with caries incidence in 12 to 30 month old children. J Dent Res.2000;79(6):1371-1377.
- Schwertner C, Moreira M, Faccini L & Hashizume L. Biochemical composition of the saliva and dental biofilm of children with Down syndrome. Int J of Paed Dent. 2016;26(2):134-140. doi: 10.1111/ipd.12168
- Topaloglu-Ak A, Ertugrul F, Eden E, Ates M, Bulut H. Effect of orthodontic appliances on oral microbiota-6 month follow-up. J Clin Pediatr Den. 2011;35(4):433-436.
- Maret D, Marchal-Sixou C, Vergnes JN, Hamel O, Georgelin-Gurgel M, Van Der Sluis L, et al. Effect of fixed orthodontic appliances on salivary microbial parameters at 6 months: A controlled observational study. J Appl Oral Sci, 2014;22(1):38-43. doi.org/10.1590/1678-775720130318